Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.


DNA/RNA synthesis and reference material extraction for RT-LAMP
Three types of positive controls were used for RT-LAMP assays: plasmids, in vitro transcripts, and reference samples. For pDrive-N1 and pDrive-RP plasmid construction, RT-PCR of the gene fragments corresponding to N1 (202 bp) and RP (232 bp) was performed using the F3/B3 primers and a positive RNA sample from previously confirmed infected individual. Super-Script™ III Platinum™ One-Step qRT-PCR Kit (Thermo Scientific, Waltham, MA, USA) was used following the manufacturer's instructions. The PCR products were cloned into the pDrive vector (Qiagen, Hilden, Germany) and verified by Sanger sequencing (Macrogen, South Korea). For in vitro transcripts, 9 μg of pDrive-N1 or pDrive-RP plasmid were linearized with BamHI-HF or HindIII-HF restriction enzymes (New England Biolabs, Beverly, MA, USA) respectively, and purified with Zymoclean Gel DNA Recovery Kit (Zymo Research, La Jolla, CA, USA). In vitro transcription was performed using 500 ng of linearized plasmid as template with the MAXIscript SP6/T7 Transcription Kit (Invitrogen, Waltham, MA, USA) according to manufacturer's instructions. An extended incubation time of 1 h for DNase digestion was used. SP6 or T7 RNA polymerase was selected to produce transcripts with the same polarity as SARS-CoV-2 genome. Transcribed products were mixed with 25 μL of a lithium chloride solution (7.5 M LiCl, 10 mM EDTA) and incubated overnight at -20˚C. RNA was recovered by centrifugation at 21,000 g for 45 min at 4˚C. The pellet was washed with 300 μL of cold 70% ethanol, centrifugated at 21,000 g for 45 min at 4˚C and resuspended in 30 μL of RNase-free water. The single stranded RNA (ssRNA) copy number was determined using Eq 1 [28]: ssRNA copy number ðmoleculesÞ ¼ X ng � 6:022 � 10 23 molecules=mol ðN � 340 g=molÞ � 1 � 10 9 ng=g ð1Þ where X is the amount (ng) of ssRNA and N is the length (bp) of ssRNA. Ten-fold serial dilutions (from 5×10 10 to 50 copies/μL) were prepared. For reference samples, 150 μL of AccuPlex SARS-CoV-2 Reference Material Kit (SeraCare, Milford, MA, USA), including reference materials and negative control, were extracted using the Viral RNA Purification Kit (Biopure, CDMX, Mexico), following the manufacturer's instructions.
All chromatographic steps were analyzed by electrophoresis through 8% tricine-SDS-PAGE gels. Bst and RT protein concentration was determined using a NanoDrop One Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and purity was evaluated by densitometry of Coomassie brilliant blue stained tricine-SDS-PAGE gels using the Image Lab 6.1 software (Bio-Rad, Hercules, CA, USA).  8,40,200 or 420 ng of recombinant RT. All reactions were evaluated by electrophoresis in 2% agarose gel, and nucleic acids produced amounts were determined by densitometric analysis using the Image Lab 6.1 software (Bio-Rad, Hercules, CA, USA) using 1 μL of 1 Kb Plus DNA Ladder (Invitrogen, Waltham, MA, USA) as standard. The total amount of nucleic acid produced was plotted against the total amount of enzyme per reaction. Then, the equation of the exponential region of the curve was obtained and used to determine the specific enzymatic activity. Bst and RT specific enzymatic activity was expressed as units per milligram (U/mg) of enzyme. One unit of Bst was defined as the amount (ng) of enzyme required to produce 100 ng of dsDNA in 30 min at 65˚C, while one unit of RT was defined as the amount (ng) of enzyme required to produce 100 ng of cDNA in 60 min at 50˚C.

Optimization assays of RT-LAMP for SARS-CoV-2 detection
The amplification temperature (60, 63 and 65˚C)  Colorimetric end-point RT-LAMP reactions were performed in a T100 TM ThermalCycler (Bio-Rad, Hercules, CA, USA) at 65˚C for 40 min. The reaction tubes were chilled on ice and color changes were documented. Amplification products were visualized by electrophoresis through 2% agarose gel. The effect of additives (40 mM GuHCl or 0.8 M betaine) was evaluated by fluorometric real-time and colorimetric end-point RT-LAMP assays as previously described using in vitro transcribed N1 gene fragment as template.

Analytical sensitivity and specificity of RT-LAMP assays for SARS-CoV-2 detection
The analytical sensitivity or limit of detection (LOD) and specificity of RT-LAMP assays were evaluated in triplicate under optimized conditions. LOD was determined with 1×10 11 to 1×10 2 copies/reaction of N1 in vitro transcript. The analytical specificity was determined by fluorometric real-time RT-LAMP assay using the NATtrol Respiratory Panel-2 (RP2) Controls, control 1 (NATROL-1) and control 2 (NATROL-2) (ZeptoMetrix, Buffalo, NY). AccuPlex SARS-CoV-2 reference material (SeraCare, Milford, MA, USA) and 1×10 9 copies of in vitro transcribed N1 gene fragment were used as positive controls.

Evaluation of the RT-LAMP assays for SARS-CoV-2 detection in clinical samples
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee for Human Research of Centro de Investigación y de Estudios Avanzados (CINVESTAV) (Protocol number: 062/2020). Written, informed consent was signed by each participant. No minor participants were included in this study.
Saliva samples were collected from 100 volunteers from people seeking testing for SARS--CoV-2 at CINVESTAV, Mexico, in September 2021 employing the Biopure Self-Sampling Kit (Biopure, CDMX, Mexico) and following the manufacturer's instructions. Collected samples were inactivated at 65˚C for 20 min. RNA extraction was performed using Viral RNA Purification Kit (Biopure, CDMX, Mexico) and quantified using a NanoDrop One (Thermo Scientific, Waltham, MA, USA). Purified RNA samples were analyzed by colorimetric end-point RT-LAMP assays. Reactions of 12.5 μL were performed with recombinant enzymes, without additives, and optimized conditions with N1 primers and RP primers. Additionally, samples were evaluated by RT-qPCR according to two different diagnostic methodologies: CDC and Berlin protocols. For CDC RT-qPCR protocol, 2019-nCoV-N1 (target: a region of the N gene) and RP (target: human RNase P) primers/probe sets were used [32]. For Berlin protocol, RdRp_SARSr (target: RNA-dependent RNA polymerase viral gene) forward and reverse primers, with RdRp_SARSr-P1 (for SARS-CoV-2, SARS-CoV and bat-SARS-related CoVs detection) or RdRp_SARSr-P2 (specific for SARS-CoV-2, will not detect SARS-CoV) probes were used [33].

Statistical analysis
To determine significant statistical differences between treatments, non-parametric analysis Kruskall-Wallis was employed to compare the means of groups. When significant differences were observed, Dunn's multiple comparisons test was performed to determine which groups were different. For real time RT-LAMP fluorometric data, we compare the time to threshold to determine statistical significance. Means and standard deviations (SD) were calculated and represented as mean ± SD. All calculations and graphics were performed with the GraphPad Prism software version 8.0.1 by setting a p < 0.05 as a significant value.

Expression and purification of recombinant Bst and RT
For recombinant protein production in E. coli carrying the pKJE7 and Bst or RT plasmids, induction conditions were selected to obtain the maximum yield of soluble protein (S2 and S3 Figs). Both enzymes were induced at 16˚C for 16 h in LB medium with 0.5 mM IPTG, recovered in the soluble fraction, and purified by Ni 2+ -IMAC chromatography (Fig 1). The biomass yield of the recombinant expression cultures for the Bst and RT enzymes were 3.89 and 4.06 g/ L, respectively. After the elution with 500 mM imidazole, recombinant Bst and RT were recovered in two and five fractions, respectively (Fig 1A and 1D). The Ni 2+ -IMAC elution fractions were desalted and a second purification step through heparin chromatography (HC) for Bst enzyme ( Fig 1B) and cation exchange chromatography (CEC) for RT ( Fig 1E) were performed. All chromatographic steps showed a single peak corresponding to the fractions containing recombinant proteins (S4 Fig). SDS-PAGE analysis of HC for Bst revealed the enzyme was obtained in at least one of the eluted fractions ( Fig 1B, Lane 4). Similarly, the second purification step for RT allowed to recover this enzyme in three elution fractions ( Fig 1E, Lane 8-10). Densitometric analysis of SDS-PAGE final formulated fractions showed that recombinant Bst was obtained at 1.6-2.0 mg/mL (purity >85%) with an estimated size of 66.6 kDa (Fig 1C). On the other hand, recombinant RT was obtained at 1.2-1.5 mg/mL (purity ranging from 80.1% to 99.2%) with an estimated size of 72 kDa (Fig 1F).

Bst and RT enzymatic activity assay
For Bst enzymatic activity assay evaluated by LAMP reactions, similar amounts of DNA were amplified with 1,072 ng and 536 ng of Bst. However, the amount of amplified DNA showed a slight decrease using 432 ng and a large decrease with 320 ng of recombinant Bst. Similar results were observed for both DNA plasmid concentrations used as template. For RT enzymatic activity assay evaluated by cDNA synthesis, 91 ng of cDNA were obtained with 420 ng and 200 ng of recombinant RT, while 96 ng of cDNA were obtained with 200 U of the commercial enzyme (SuperScript III RT, Invitrogen). The amount of synthesized cDNA decreased using 40 ng of recombinant RT and it was not detectable when 8 ng of RT were used (S5 Fig). The calculated specific enzymatic activity was 2.99×10 3 U/mg for recombinant Bst and 4.15×10 3 U/mg for RT.

Optimization assays of RT-LAMP for SARS-CoV-2 detection
To evaluate temperature effect, fluorometric real-time LAMP reactions were also performed. The N1 primer set showed a more efficient amplification at 65˚C with statistically significant difference (Fig 2A). Subsequently, real-time RT-LAMP assays under different Tris-HCl concentrations (0 to 1 mM) and two pH values (8.5 and 8.8) were performed ( Fig  2B). Tris-HCl concentration at different pH had little effect on the amplification of in vitro transcribed N1 RNA fragment, since only 1 mM Tris-HCl, pH 8.5 and absence of Tris in a pH 8.8 buffer showed statistically significant differences between treatments. For this reason, reaction buffer without Tris-HCl at pH 8.8 was selected as optimal for the fluorometric RT-LAMP assay (Fig 2B). On the other hand, the selected conditions were tested in colorimetric end-point RT-LAMP reactions. In absence of Tris-HCl (pH 8.8) an evident visual detection of color transition from pink-red (negative reaction) to yellow (positive reaction) was obtained. These conditions were also established as the optimal for the colorimetric RT-LAMP assay (Fig 2C).

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Detecting SARS-CoV-2 in site For these reasons, GuHCl or betaine were not included as additives on RT-LAMP assays.

Analytical sensitivity and specificity of RT-LAMP assays for SARS-CoV-2 detection
The analytical sensitivity of fluorometric real time and colorimetric end-point RT-LAMP assays was evaluated under optimized conditions. Amplification curves of serial dilutions of in vitro N1 transcript showed that fluorometric real time RT-LAMP assay was able to detect 10 2 copies/reaction (Fig 3A). Analytical sensitivity analysis of colorimetric end-point RT-LAMP showed evident change color until 1x10 2 copies/reaction (Fig 3B). For analytical specificity, respiratory controls (NATROL-1 and NATROL-2) were evaluated by fluorometric real time RT-LAMP without evident amplification (Fig 3C).

Evaluation of the RT-LAM-P assays for SARS-CoV-2 detection in clinical samples
Finally, considering that colorimetric end-point RT-LAMP is a feasible alternative for POC diagnostics without the need for specialized equipment, we compared the performance of this assay with CDC and Berlin protocol-based methodologies (RT-qPCR). RNA extraction from 100 saliva samples from volunteers was performed. According to RP primers/probe set from CDC RT-qPCR protocol, internal control was detected in all samples with Ct values ranging from 24.90-31.93, and 59% were positive to N gene detection with Ct values of 17.71-38.70 (S1 File). Moreover, Berlin RT-qPCR protocol-based methodology using RdRp_SARSr-P2 probe resulted in 34% positive detection of SARS-CoV-2 with Ct values ranging from 16.68-37.16, and 38% positive detection of SARS-CoV-2, SARS-CoV and bat-SARS-related CoVs (Ct values from 19.77-39.31) using RdRp_SARSr-P1 probe in saliva samples. On the other hand, colorimetric end-point RT-LAMP resulted in 26% positive detection for SARS-CoV-2. Comparing RT-LAMP assay to RT-qPCR Ct values, sensitivity (defined as the ability of a screening test to detect a positive result, being based on the positive result rate [34] detected by a reference method) of RT-LAMP compared with CDC protocol-based Ct values below 30 was 100% (Table 1). However, RT-LAMP sensitivity decreased in samples with Ct values until 35 (68.4%) and performed poorly (44.1% sensitivity) in samples with Ct > 35. Out of 41 negative CDC results, none were observed to show a positive reaction when analyzed with RT-LAMP. Hence a 100% specificity (defined as the ability of a screening test to detect a negative result, being based on the negative result rate [34] detected by a reference method) was obtained for colorimetric RT-LAMP. Targeting SARS-CoV-2, the sensitivity of RT-LAMP compared with Berlin protocol-based Ct values until 30 was 100% but decreased to 81.3% and 76.5% for Ct > 30 and Ct > 35, respectively. Out of 66 Berlin protocol-based negative results, none showed a positive RT-LAMP reaction. Therefore, 100% specificity was obtained for colorimetric assay. These results suggested that colorimetric RT-LAMP assay developed with recombinant enzymes can be used for a sensitive and specific detection of SARS-CoV-2 in complex samples such as RNA purified from saliva (Fig 4).

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Detecting SARS-CoV-2 in site

Discussion
The first step in managing COVID-19 is the rapid and accurate detection of SARS-CoV-2 enabled by identification of viral targets [35]. RT-LAMP is a nucleic acid detection technique that has become an excellent alternative for POC testing due to its characteristics of a rapid, simple, sensitive, and specific method [12][13][14]. Nevertheless, most of the reported assays for the detection of SARS-CoV-2 by RT-LAMP use commercial enzymes or reaction buffers subject to availability. In this work, RT-LAMP diagnostic tests (fluorometric real time assay and colorimetric end-point assay) were developed with emphasis in the production and purification of the enzymes necessary to implement the technique. For this purpose, Bst DNA polymerase and MM4-RT were expressed and purified. Previous studies have reported a high catalytic activity and thermostability for both enzymes [30,36] which make them suitable to be used in RT-LAMP assays. Additionally, attending the high demand for diagnostic testing against SARS-CoV-2, we do not discard the possibility of using plasmids encoding for other DNA polymerases (Bsm, Bst2.0, Bst3.0 or Bst LF) or thermostable reverse transcriptases such as AMV or HIV retrotranscriptases [37,38].
During the purification of the Bst enzyme, the heat clarification step allowed the elimination of a high proportion of non-thermostable proteins from E. coli [39], this step favored protein-resin interaction and protein recovery in the first purification by Ni 2+ -IMAC column. The second step by heparin chromatography, commonly used for the purification of proteins with affinity to nucleic acids [40], allowed the recovery of the active enzyme with high purity. Similarly, the purification process used for the RT enzyme allowed the protein to be recovered with high purity. The purification workflow proposed in this work for Bst and RT could be used to produce enzymes for the development of RT-LAMP-based detection tests.
RT-LAMP assays included six primers targeting SARS-CoV-2 nucleocapsid gene sequence. ORFs located in the 3' region of viral genomes are usually better candidates for the development of detection tests against single-stranded RNA viruses, such as SARS-CoV-2 [41,42]. Additionally, the 5' region of N1 gene overlaps with ORF9b, which explains high conservation and low mutation rate [43,44]. Nucleocapsid gene is present in the genomic RNA and in nine control. M: DNA molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen). N1 transcript: 1×10 9 copies of in vitro transcribed N1 gene. Reference material: 2 μL of AccuPlex SARS-CoV-2 reference material.
https://doi.org/10.1371/journal.pone.0279681.g003 of the subgenomic RNA molecules of SARS-CoV-2, inferring the virus could be detected in all these molecules (Fig 1A). Bioinformatic analysis demonstrated that the primers matched 100% the N gene of the alpha, beta and delta SARS-CoV-2 variants of concern, while one nucleotide mismatch was found for the omicron variant. This suggests that RT-LAMP assays have a broad capacity to accurately detect several SARS-CoV-2 variants. Primer concentration is critical for RT-LAMP reactions as an inadequate amount could decrease the sensitivity of the assay, and consequently could affect the reliability of the detection test [45]. In this study, a low primer concentration for N1 and RP primer sets showed an appropriate performance as no by-product formation was observed and the amplification efficiency of the target gene was not affected. A decrease in the total ion concentration (Tris-HCl, KCl, MgCl 2 ) has been shown to affect reaction efficiency [46]. Our results indicated that buffer composition for fluorometric realtime and colorimetric end-point RT-LAMP assays was adequate, as optimal enzymatic activity, and a high reaction efficiency were obtained. Both assays could be carried out in absence of Tris-HCl pH 8.8 without affecting the reaction, even the color transition in the case of colorimetric detection method. Although it has been reported that additives such as guanidine hydrochloride (GuHCl) [47,48] and betaine [45,49] can improve the speed and sensitivity of RT-LAMP reactions. However, we did not detect statistical differences after betaine addition and GuHCl interfered with visual detection. Previous reports indicated variable and stochastic results with RT-LAMP assays when less than 100 copies of synthetic viral RNA standard per reaction were used [37,47]. Instead, a LOD equal or higher than 1000 copies per reaction resulted in a robust detection of the target gene [37]. Therefore, considering the LOD obtained in this work, it would be expected a sensitive and robust detection of SARS-CoV-2 by both developed RT-LAMP assays. A common strategy to achieve lower LOD than those obtained here is to use two or more target genes in the same reaction [50]. In addition, the detection of SARS-CoV-2 N gene by RT-LAMP using the selected primers showed to be highly specific as no cross-reactivity was observed with several common respiratory pathogens including influenza virus isolates (AH1, AH1N1, AH3 and B), parainfluenza (type 1, 1A, 2 and 4), adenovirus (type 1, 3 and 31), Mycoplasma pneumoniae, human Metapneumovirus, Legionella pneumophila, Chlamydophyla pneumoniae, Bordetella pertussis, Bordetella parapertussis, RSV type A, respiratory syncytial virus A, and other coronaviruses (NL63, OC43, HKU-1, 229E).

Protocol-based methodology
The developed colorimetric RT-LAMP assay was subjected to a clinical evaluation for SARS-CoV-2 detection in saliva samples. A high rate of false positives has been reported in colorimetric RT-LAMP assays using phenol red caused by the high pH variability of saliva samples. Indeed, the pH range of saliva (6.8 to 7.4) is close to the pH transition zone of phenol red (pH 7.5) [17,51,52]. We employed saliva instead of conventional nasopharyngeal swabs samples since discomfort, coughing and sneezing are usually produced during sample collection [53]. In addition, this stage can represent a critical moment of exposure to the virus in healthcare professionals [54]. Saliva samples possess high heterogenicity [55,56], low yields during RNA extraction [56,57], presence of polymerase inhibitors [58] and genetic material from the oral microbiome [59]. However, RT-LAMP is more resistant to inhibitors than RT-qPCR [60], and saliva samples are especially useful for screening asymptomatic patients or those with mild COVID-19 infection [61]. Furthermore, it has recently been described as the ideal biological fluid for the detection of strains with tropism towards the upper respiratory tract (such as the omicron variant) [62].
Developed colorimetric end-point RT-LAMP assay using the recombinant Bst and RT enzymes was able to amplify the human RNase P gene in all samples that were positive for this internal reaction control by CDC RT-qPCR test (S1 File). Considering previous reports of different RT-LAMP procedures compared with several RT-qPCR reference protocols, sensitivity for SARS-CoV-2 detection is nearly 100% for Ct values until 30 using distinct viral targets in pharyngeal swab samples [63,64]. Ct values close to 30 have been reported as cut-off in RT-qPCR assays compared with LAMP for the detection of SARS-CoV-2 [20,65] due to they are related with low viral load samples and they are close to the detection limit for viral culture with probably scarce epidemiological significance [66][67][68]. Other studies have shown that Ct values > 30 are useful predictors of low infectivity, low risk of intubation, and low mortality [69,70], and this has been used as hospital discharge criteria [71,72]. Indeed, detection of SARS-CoV-2 by colorimetric end-point RT-LAMP assay developed in this work demonstrated a 100% sensitivity among samples with Ct < 30 when RT-qPCR CDC or Berlin diagnostic methodologies were used as reference protocols. However, considering CDC and Berlin Ct values up to 35 and 40, sensitivity of our developed assay decreased. It has been reported that clinical samples with Ct values > 30 (corresponding to low viral load) made the detection process quite challenging [73]. Even RT-qPCR assays could detect reliably specimens with Ct values < 30, but did not detect 40-60% of specimens with Ct � 30 [73]. In this regard, we observed a discrepancy between CDC and Berlin positive results (59% and 34%, respectively) using RNA extracted from saliva samples (Table 1). According to a previous study, four WHO approved RT-PCR diagnostic protocols for SARS-CoV-2 (including those used in our study) showed discordant results in almost 30% of cases evaluated [74]. Additionally, Berlin RdRp-SARSr primers-probe set has been reported to show low sensitivity compared with other primers-probe sets [75]. Finally, 100% specificity was obtained for our colorimetric end-point RT-LAMP assay since no false positive reactions were observed in comparison with RT-qPCR CDC or Berlin reference protocols.
The main advantage of our method is that it is fully independent on commercial suppliers (enzymes or buffers), that could be subjected to limited availability due to the ongoing pandemic and necessity of continuous diagnostic test. Our development of RT-LAMP assays using recombinant enzymes and in-house-made buffers, together with other efforts [76,77], confirmed the possibility for the implementation of broad inexpensive testing especially in areas where availability is restricted by economic or supply/demand issues.

Conclusions
The purified recombinant Bst DNA polymerase and MM4-RT were used to develop fluorometric real time and colorimetric end-point RT-LAMP assays to detect the SARS-CoV-2 N gene. Colorimetric end-point RT-LAMP reactions without Tris-HCl or additives and with a low primer concentration were used successfully to detect SARS-CoV-2 in RNA isolated from saliva samples from infected individuals and to discriminate from negative samples. Fluorometric real time RT-LAMP represents a sensitive and efficient technique for quantifying viral loads, while colorimetric end-point RT-LAMP is an in-expensive and high throughput POC assay independent of sophisticated equipment.